Delivery of biologically active polypeptides

ABSTRACT

Biologically active polypeptides and/or antigens are delivered by administering to a subject a non-invasive or non-pathogenic bacterium which expresses one or more antigens or polypeptides. The non-invasive or non-pathogenic bacterium can be included in delivery systems or pharmaceutical formulations.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation of application Ser. No.09/060,878, filed Apr. 16, 1998, pending, which is a §371 ofPCT/GB96/02580 filed on Oct. 21, 1996.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to the delivery of biologicallyactive polypeptides in vivo. In particular, it relates to the use ofnon-invasive bacteria, generally Gram-positive bacteria such asLactococcus, in providing biologically active polypeptides in the body,especially at mucosa. In one aspect, this relates to the provision of anadjuvant effect by means of which an immune response raised to anantigen is enhanced. Nucleic acid constructs and host organisms forthese applications are also provided.

[0003] The limited number of adjuvants approved for use in humanvaccines (owing to the toxicity or pathogenicity of the most activeagents such as Freund's complete adjuvant) and the discovery during thepast 20 or more years of numerous polypeptides involved in theproliferation, differentiation and activation of B cells and T cells hasdrawn attention to the possibility of using these factors (cytokines) toaugment responses to vaccines and to direct the immune response to aparticular vaccine along desired pathways. The need for this approachhas become even more apparent as recent immunological discoveries haveemphasized that cell-mediated and antibody-mediated immune responsesare, to a large degree, mutually exclusive responses. Whether antibodyformation or effector T cells and macrophages are activated isdetermined by which a particular array of cytokines is elicited by anygiven antigen, pathogen or vaccine. Most important is the functionalactivity of the types of helper T cells, TH1 or TH2, which are involvedin the response to any particular antigen or invading pathogen.

[0004] Since protective immunity to a pathogenic agent usually arises asa consequence either of antibody formation (extracellular pathogens,soluble toxins or intracellular pathogens following their release intotissue fluids from dead, dying or productive cells) or of cell-mediatedresponses (intracellular pathogens), it is, in principle, highlyadvantageous to be able to direct immune responses to a vaccine towardseither antibody formation or T cell and macrophage activation. In orderthat the protective effects of vaccination should persist for as long aspossible, it is also important to be able to enhance the amplitude,duration and memory components of the immune response.

[0005] For these reasons, numerous investigators have focused theirattention on the possibility of harnessing one or more of the members ofthe cytokine network of signaling proteins as vaccine adjuvants. Thisapproach may be even more significant when it is considered that theloss of helper T cells—and hence of their cytokine output—may beassociated with the failure of individuals suffering from certain typesof inherited or acquired immunodeficiencies to be able to respond toparticular vaccines.

[0006] Although much attention has been paid to the use of cytokines forthese purposes, only limited success has been reported in harnessingcytokines as adjuvants. Considerable difficulty has been encountered inadministering adjuvant cytokines by methods which would be appropriatefor inclusion in a vaccine regimen. This difficulty may be exemplifiedby reference to studies of the use of Interleukin-2 (IL-2) as anadjuvant.

[0007] IL-2 has attracted particular attention as a possible adjuvantbecause, although its principal source is thought to be T helper 1cells, its major activities are believed to include involvement in wideranging aspects of immune responses, such as T cell proliferation, thesynthesis of other cytokines, B cell growth and immunoglobulinsynthesis. Thus, IL-2 is a T cell-derived cytokine which was firstdescribed as a T cell growth factor. It is now known to stimulate growthand differentiation of T cells, B cells, NK cells, monocytes,macrophages and oligodendrocytes. In general, adjuvant activity on thepart of IL-2, which has been reported by many workers, has been found todepend on the use of multiple injections of the cytokine or itsincorporation into liposomes or oily emulsions. To avoid this need,other workers have either coexpressed IL-2 with vaccine antigens inrecombinant bacterial and viral vectors or have engineered IL-2 antigenfusion proteins; the latter are claimed to provide marked enhancement ofthe immunogenicity of the antigenic component of the fusion partner.

[0008] Other desirable characteristics of vaccines include the need tobe as innocuous as possible, to act effectively following theadministration of the smallest possible number of doses, and to besuitable for administration via mucosal surfaces (e.g., orally,intranasally, or intra-vaginally), thus obviating the need forhypodermic needles, and activating local, mucosal immune responses inaddition to systemic immune responses. The capacity for continuedproliferation of live, attenuated pathogens has resulted in numerousstudies of the use of recombinant vaccine strains of viruses andbacteria (such as vaccine strains of pox viruses, or of salmonella andtubercle bacteria) as agents for the delivery of heterologous antigens.

[0009] We have previously developed systems for the expression ofheterologous antigens in the non-pathogenic, non-colonizing,non-invasive food-grade bacterium Lactococcus lactis (see UK patentGB-2278358B). We have shown previously that Lactococcus lactis is ableto produce and secrete biologically active murine IL-2 when cultured invitro (Steidler et al., Applied and Environmental Microbiology, April1995, Vol. 61, No. 4, pp1627-1629). However, owing to the fact thatLactococcus lactis is non-invasive—it is indeed not a commensalbacterium nor otherwise normally associated with the colonization ofmucosal surfaces in animals—it was not obvious that this bacterium couldbe successfully employed in a vaccination strategy which required theformation of an adjuvant cytokine in vivo. We have previously shown(GB-2278358B) that heterologous antigen can be fully antigenic whenaccumulated within the cytoplasm of Lactococcus lactis (from which it ispresumed to leak in vivo as the cells are digested by phagocytic cells).

[0010] By the manipulation of the appropriate genetic elements, we haveprovided nucleic acid constructs (here artificial operons—coordinatelytranscribed multigene units) for coexpression in Lactococcus lactis ofan antigenic polypeptide (exemplified here using tetanus toxin fragmentC-TTFC) and a biologically active cytokine polypeptide (exemplified hereusing Interleukin 2 (IL-2) and also Interleukin-6 (IL-6).

[0011] The IL-6 cytokine has been shown by other workers to have thecapacity to augment murine antigen-specific antibody responses in vivoand in vitro, and we have also been able to prepare expression units forIL-6 in L. lactis. IL-6 is a multi-functional cytokine secreted by bothlymphoid and non-lymphoid cells which is known to possess pleiotropicactivities that play a central role in host defense. IL-6 can exertgrowth-inducing, growth-inhibitory and differentiation-inducingactivities, depending on the target cells. These activities includedifferentiation and/or activation of T cells and macrophages, growthpromotion of B cells (seen as growth—promotion of B cell tumor lines invitro), terminal differentiation (secretion of immunoglobulins) in Bcells, and—acting systemically—elicitation of the hepatic acute-phaseprotein response. Most importantly for the purposes of mucosalimmunization, IL-6 has been shown to induce high-rate IgA secretion inIgA-committed B cells.

SUMMARY OF THE INVENTION

[0012] To exemplify the present invention, operons for IL-2 and IL-6coexpression were separately constructed in a constitutive expressionvector (pTREX1, also known as pEX1) so that the transcription of theTTFC gene and the interleukin gene could be controlled by the activityof a lactococcal promoter element of previously defined activity(so-called P1). The constructs were prepared so that, followingtranslation of the mRNA transcribed from the artificial operons, theTTFC antigen would accumulate intracellularly.

[0013] When preparations of these bacteria were administeredintranasally to mice, bacteria engineered to express either IL-2 or IL-6elicited approximately 10× more anti-TTFC antibody than the constructswhich expressed the TTFC alone. Thus, either of these interleukinspossessed distinctive adjuvant activity in the experimental system.

[0014] It was not obvious from either the capacity of Lactococcus lactisto deliver a heterologous antigen or its ability to produce IL-2 invitro that it would be an appropriate vehicle for a delivery of acytokine in vivo such that sufficient, active cytokine would be providedto provide an adjuvant effect. Lactococcus lactis is non-invasive andnon-colonizing, which means that when these bacteria are used to deliveran antigen to the immune system, e.g., via a mucosal surface, they aremost likely to enter lymphoid tissue as a consequence of phagocytosis bythe M (or microfold) cells which sample the contents of mucosalsecretions adjacent to mucosal lymphoid tissue. Microparticulateantigens (e.g., tetanus toxoid incorporated into poly L-lactidemicroparticles) enter lymphoid tissue passively in this way, whereaspathogenic bacteria (or attenuated vaccines) such as species ofListeria, Salmonella and Shigella are able to invade cells and tissuesby actively stimulating their uptake into mucosal epithelial cells, inaddition to gaining entry via M cells. Since the activity of cytokinesas adjuvants has been found previously to require multiple injections orsustained-release delivery (Heath and Playfair (1992), Vaccine 7:427-434), and since the cytokines will only be protected fromproteolytic digestion within phagocytic cells while the Lactococcuslactis cells remain intact or viable, it is unexpected that lactococcalcells expressing cytokines should display marked adjuvant activity asdemonstrated herein. This can perhaps be appreciated if it is understoodthat death and dissolution of the bacterial particles will favor antigenrelease but prevent more than very transient production of cytokines.Nevertheless, our findings indicate that the expression of IL-2 or IL-6by Lactococcus lactis does have a marked adjuvant effect. Even if theexpressor bacteria were to be administered by a parenteral rather than amucosal route the same considerations would apply.

[0015] Thus, since Lactococcus lactis is not invasive—indeed it is not acommensal bacterium and it also depends for its nutrition on theprovision of amino acids and peptides which are unlikely to be availablein vivo—the demonstration that the cytokine-secreting strains of L.lactis are nevertheless able to augment antibody production issurprising. Hence, these results demonstrate for the first time thatrecombinant strains of Lactococcus lactis can be used to synthesize anddeliver biologically active molecules in vivo. Of particular interest isthe fact that these results demonstrate the feasibility of augmentingthe mucosal as well as the systemic immune response since IL-6 has beenshown to be a cytokine able to induce a high rate of IgA secretion inIgA-committed B cells.

[0016] The finding that Lactococcus lactis is able to sustain itsbiological activity on a mucous membrane for a sufficient length of timeto deliver a biologically active dose of either of two differentrecombinant cytokines and thereby augment an immune response to aheterologous antigen demonstrates broad applicability for the deliveryof polypeptides for purposes other than adjuvant activity alone.

[0017] The capacity of L. lactis to produce and secrete polypeptidesdemonstrates that it is possible to utilize these bacteria for in vivoproduction and delivery of polypeptides which are known to be active atmicromolar, nanomolar or picomolar concentrations. Since precise dosingof these polypeptides and the need for the coincidental introduction ofbacterial cells are of lesser concern for veterinary than humanapplications, it is likely that this method for delivering recombinantpolypeptides will be especially valuable in veterinary applications.However, even within human medicine, the fact that cytokine output canbe constrained to the sites of deposition of harmless bacterial cellsand is available close to the antigen during the earliest phases of theimmune response may favor its use in circumstances—such as adjuvantactivity—where the biologically active polypeptide is best localized inorder to avoid toxic systemic side effects.

[0018] Thus, the present invention provides:

[0019] (i) a method of delivering one or more biologically activepolypeptides which comprises administering to a subject a non-invasiveor non-pathogenic bacterium which expresses the one or morepolypeptides;

[0020] (ii) a method of delivering one or more antigens which comprisesadministering to a subject a non-invasive or non-pathogenic bacteriumwhich expresses the one or more antigens; and

[0021] (iii) a method of delivering one or more antigens and/or one ormore biologically active polypeptides which comprises administering to asubject a non-invasive or non-pathogenic bacterium which expresses boththe one or more antigens and the one or more heterologous biologicallyactive polypeptides.

[0022] The biologically active polypeptides can be either homologous tothe bacterium or heterologous, derived from either eukaryotic sources orprokaryotic sources, or their viruses.

[0023] According to another aspect of the present invention, there isprovided a non-invasive or non-pathogenic bacterium expressing (i) oneor more heterologous biologically active polypeptides and (ii) one ormore antigens.

[0024] “Biological activity” refers to the ability to perform abiological function and, with reference to a polypeptide, implies thatthe polypeptide adopts a stable conformation (“folded form”) which isthe same as or closely analogous to its native configuration. Whenfolded correctly or substantially correctly, for example, with formationof proper folded units, α-helices, β-sheets, domains, disulphidebridges, etc., a polypeptide should have the ability to perform itsnatural function. Generally, the unit of function in a polypeptide is adomain.

[0025] The mere ability to be bound by an antibody or other receptor,either with or without elicitation of an immune response, is passive anddoes not constitute “biological activity.” Any antigen has the abilityto be bound by an antibody but is not necessarily biologically active.

[0026] A “heterologous” polypeptide is one not native to the bacterium,i.e., not expressed by the bacterium in nature or prior to introductioninto the bacterium, or an ancestor thereof, of encoding nucleic acid forthe polypeptide.

[0027] A bacterium according to the present invention will, in general,be Gram-positive and may, in principle, be any innocuous bacterium, forexample, Listeria innocua, Slaphylococcus xylosus or a Lactococcus.Lactococci, in particular Lactococcus lactis, represent a preferredembodiment of the present invention. Such bacteria are non-colonizing.

[0028] The skilled person will appreciate that the methods of thepresent invention could be used to deliver a range of biologicallyactive polypeptides. Examples of suitable polypeptides include oneswhich are capable of functioning locally or systemically, e.g., is apolypeptide capable of exerting endocrine activities affecting local orwhole-body metabolism. The biologically active polypeptide(s) is/areone(s) which is/are capable of regulating the activities of cellsbelonging to the immunohemopoietic system and/or the one or morebiologically active polypeptides is/are one(s) which is/are capable ofaffecting the viability, growth and differentiation of a variety ofnormal or neoplastic cells in the body or affecting the immuneregulation or induction of acute phase inflammatory responses to injuryand infection and/or the one or more biologically active polypeptidesis/are one(s) which is/are capable of enhancing or inducing resistanceto infection of cells and tissues mediated by chemokines acting on theirtarget cell receptors, or the proliferation of epithelial cells or thepromotion of wound healing and/or the one or more biologically activepolypeptides modulates the expression or production of substances bycells in the body.

[0029] Specific examples of such polypeptides include insulin, growthhormone, prolactin, calcitonin, luteinizing hormone, parathyroidhormone, somatostatin, thyroid-stimulating hormone, vasoactiveintestinal polypeptide, a structural group 1 cytokine adopting anantiparallel 4α helical bundle structure such as IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, GM-CSF, M-CSF, SCF, IFN-γ,EPO, G-CSF, LIF, OSM, CNTF, GH, PRL or IFNα/β, a structural group 2cytokine which is often cell-surface associated, form symmetrichomotrimers and the subunits that take up the conformation of β-jellyroll described for certain viral coat proteins such as the TNF family ofcytokines, e.g., TNFα, TNFβ, CD40, CD27 or FAS ligands, the IL-1 familyof cytokines, the fibroblast growth factor family, the platelet-derivedgrowth factors, transforming growth factor β and nerve growth factors, astructural group 3 cytokine comprising short chain α/β molecules, whichare produced as large transmembrane precursor molecules which eachcontain at least one EGF domain in the extracellular region, e.g., theepidermal growth factor family of cytokines, the chemokinescharacterized by their possession of amino acid sequences grouped aroundconserved cysteine residues (the C—C or C—X—C chemokine subgroups) orthe insulin-related cytokines, a structural group 4 cytokine whichexhibits mosaic structures such as the heregulins or neuregulinscomposed of different domains, e.g., EGF, immunoglobulin-like andkringle domains.

[0030] Alternatively, the biologically active polypeptide can be areceptor or antagonist for biologically active polypeptides as definedabove.

[0031] The bacterium expresses the biologically active polypeptide andthe antigen from nucleic acid contained within it. The nucleic acid maycomprise one or more nucleic acid constructs in which nucleic acidencoding the biologically active polypeptide and nucleic acid encodingthe antigen are under the control of appropriate regulatory sequencesfor expression in the bacterium.

[0032] Suitable vectors comprising nucleic acid for introduction intobacteria can be chosen or constructed containing appropriate regulatorysequences, including promoter sequences, terminator fragments, enhancersequences, marker genes and other sequences as appropriate. Vectors maybe plasmids, viral, e.g., phage or phagemid, as appropriate. For furtherdetails see, for example, Molecular Cloning. a Laboratory Manual: 2ndedition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press.Many known techniques and protocols for manipulation of nucleic acid,for example, in preparation of nucleic acid constructs, mutagenesis,sequencing, introduction of DNA into cells and gene expression, andanalysis of proteins, are described in detail in Short Protocols inMolecular Biology, Second Edition, Ausubel et al. eds., John Wiley &Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. areincorporated herein by reference.

[0033] In a preferred embodiment, the coding sequences for thebiologically active polypeptide and the antigen are contained in anoperon, i.e., a nucleic acid construct for multi-cistronic expression.In an operon, transcription from the promoter results in an mRNA whichcomprises more than one coding sequence, each with its own suitablypositioned ribosome binding site upstream. Thus, more than onepolypeptide can be translated from a single mRNA. Use of an operonenables expression of the biologically active polypeptide and theantigen to be coordinated.

[0034] In an alternative embodiment, the coding sequences for thebiologically active polypeptide and the antigen are part of the samenucleic acid vector, or separate vectors, and are individually under theregulatory control of separate promoters. The promoters may be the sameor different.

[0035] A nucleic acid construct or vector comprising a coding sequencefor a biologically active polypeptide and a coding sequence for anantigen wherein each coding sequence is under the control of a promoterfor expression in a non-invasive bacterium (as disclosed—especially anon-commensal and/or non-colonizing bacterium, e.g., a Lactococcus),whether as an operon or not, is provided by a further aspect of thepresent invention.

[0036] A promoter employed in accordance with the present invention ispreferably expressed constitutively in the bacterium. Use of aconstitutive promoter avoids the need to supply an inducer or otherregulatory signal for expression to take place. Preferably, the promoterdirects expression at a level at which the bacterial host cell remainsviable, i.e., retains some metabolic activity, even if growth is notmaintained. Advantageously then, such expression may be at a low level.For example, where the expression product accumulates intracellularly,the level of expression may lead to accumulation of the expressionproduct at less than about 10% of cellular protein, preferably about orless than about 5%, for example, about 1-3%. The promoter may behomologous to the bacterium employed, i.e., one found in that bacteriumin nature. For example, a Lactococcal promoter may be used in aLactococcus. A preferred promoter for use in Lactococcus lactis (orother Lactococci) is “P1” derived from the chromosome of Lactococcuslactis (Waterfield N. R.; Le Page, R. W. F.; Wilson P. W. and Wells J.M., Gene, 165:9-15, 1995, the sequence of which is shown in thefollowing (SEQ ID NO. 1): GATTAAGTCA TCTTACCTCT TTTATTAGTT TTTTCTTATAATCTAATGAT AACATTTTTA TAATTAATCT ATAAACCATA TCCCTCTTTG GAATCAAAATTTATTATCTA CTCCTTTGTA GATATGTTAT AATACAAGTA TC

[0037] The nucleic acid construct or constructs may comprise a secretorysignal sequence. Thus, in a preferred embodiment, the nucleic acidencoding the biologically active polypeptide may provide for secretionof the biologically active polypeptide (by appropriately coupling anucleic acid sequence encoding a single sequence to the nucleic acidsequence encoding the polypeptide). The ability of a bacterium harboringthe nucleic acid to secrete the polypeptide may be tested in vitro inculture conditions which maintain viability of the organism.

[0038] Suitable secretory signal sequences include any of those withactivity in Gram-positive organisms such as Bacillus, Clostridium andLactobacillus. Such sequences may include the α-amylase secretion leaderof Bacillus amyloliquefaciens or the secretion leader of theStaphylokinase enzyme secreted by some strains of Staphylococcus, whichis known to function in both Gram-positive and Gram-negative hosts (see“Gene Expression Using Bacillus,” Rapoport (1990) Current Opinion inBiotechnology 1:21-27), or leader sequences from numerous other Bacillusenzymes or S-layer proteins (see pp341-344 of Harwood and Cutting,“Molecular Biological Methods for Bacillus,” John Wiley & Co. 1990). ForLactococcus, the leader sequence of the protein designated Usp45 may bepreferred (SEQ ID NO. 2): ATG AAA AAA AAG ATT ATC TCA GCT ATT TTA ATGTCT met lys lys lys ile ile ser ala ile leu met ser ACA GTG ATA CTT TCTGCT GCA GCC CCG TTG TCA GGT thr val ile leu ser ala ala ala pro ley sergly GTT TAC GCT val tyr ala

[0039] However, it may be preferable that the antigen accumulatesintracellularly. As discussed, preferably the level of accumulationshould allow the bacterium to remain viable, i.e., retain some metabolicactivity, and may be less than about 10% of cellular protein, preferablyabout or less than about 5% of cellular protein.

[0040] The antigen may, in principle, be any peptide or polypeptide towhich a receptor of the immune system, such as an antibody, can bind. Ina preferred embodiment, the antigen is a bacterial toxoid form of atoxin or an antigenic fragment thereof. For good compatibility ofexpression in Lactococcus, which has a bias towards A/T usage over G/Cin its coding sequences (60% A/T), the antigen may be one whose codingsequence is A/T rich (has a higher A/T content than G/C). For instance,the antigen may be a toxoid (or an antigenic fragment thereof), oranother immunogenic component from Clostridium or Pneumococcus or otherStreptococcus species. Clostridial coding sequences, for example, oftenhave >70% A/T base pair content, as do genes from the important humanmalarial parasites belonging to the genus Plasmodium.

[0041] For use in enhancing an immune response to the antigen, i.e.,antigenic peptide or polypeptide, as discussed herein, the biologicallyactive polypeptide preferably has cytokine activity. Cytokines arediscussed in “The Cytokine Facts Book,” Callard and Gearing (1994),Academic Press. Preferred polypeptides with cytokine activity areinterleukins, including Interleukin-2 (IL-2) and Interleukin 6 (IL-6).Many cytokines contain a disulphide bridge and all are secreted from thecells which naturally produce them. The reducing nature of the cytoplasmof bacterial cells would be expected to prevent formation of disulphidebridges. It would not be obvious that a polypeptide which is naturallysecreted, especially one which naturally contains a disulphide bridge,would be biologically active when retained in a bacterial cell.

[0042] Thus, in one embodiment, the biologically active polypeptide isone which is secreted from cells which naturally produce it.

[0043] The use of a cytokine to enhance an immune response to theantigen in accordance with the present invention is particularlyapposite for antigens of low immunogenicity. Furthermore, application ofan immunogen to a mucosal membrane generally elicits an IgA response.The ability of a vaccine to elicit a good (protective level) mucosalimmune response is a highly desirable feature, since it is now knownthat sIgA antibodies play a vital role in protecting mucosal surfacesagainst infection. For example, sIgA which binds to the surface of thecholera bacillus has been shown to be capable of preventing experimentalcholera in mice. sIgA, which effectively neutralized, HIV-1 may play animportant role in protecting against infection with this virus, sinceonce the virus has gained access to the body, a lifelong infection isestablished. Methods for the reliable and long-lasting induction ofmucosal sIgA responses are therefore much sought after, since the greatmajority of human viruses and bacterial pathogens initiate infections bycolonizing mucosal surfaces.

[0044] Thus, antigens of low immunogenicity from a parasite againstwhich an enhanced IgA response is beneficial may be employedparticularly advantageously in the present invention, for instance, theP28 immunogen (glutathione-S-transferase) of Schistosoma mansoni.

[0045] To generate a bacterium according to the present invention,nucleic acid is introduced into a bacterial host cell. Thus, a furtheraspect of the present invention provides a method comprising introducingnucleic acid as disclosed into a non-invasive bacterium, preferably aGram-positive bacterium and most preferably a non-commensal,non-colonizing bacterium (such as Lactococcus). The introduction mayemploy any available technique. For bacterial cells, suitable techniquesmay include calcium chloride transformation, electroporation andtransfection using bacteriophage.

[0046] The introduction may be followed by causing or allowingexpression from the nucleic acid, e.g., by culturing host cells underconditions for expression of the gene. Growing the cells in cultureunder conditions for expression of the biologically active polypeptideand the antigen may be employed to verify that the bacteria contain theencoding nucleic acid and are able to produce the encoded material.

[0047] In a further aspect, the present invention provides a method ofdelivering a biologically active dose of a polypeptide in vivo, themethod comprising administering to an individual a non-invasivebacterium containing nucleic acid for expression of a biologicallyactive polypeptide heterologous to the bacterium. As discussed supra,preferred bacteria include Lactococci such as Lactococcus lactis and apreferred route of administration may be by application to mucosa.

[0048] Although, it has previously been shown possible to express insuch bacteria a heterologous polypeptide in a biologically active form,this has only ever been done in vitro in culture conditions which areoptimized for bacterial viability and growth. In vivo, for instance, onthe mucosal membrane, the bacteria are in an environment which would notbe expected to support their growth or viability. It is thus surprisingthat such bacteria are able to deliver a polypeptide in a dose (amount)which is sufficient for the biological activity of the polypeptide toresult in a detectable biological effect.

[0049] In a preferred embodiment, the biologically active polypeptidehas cytokine activity and the bacterium may also express an antigen.Interleukins such as IL-2 and IL-6 may advantageously be delivered.

[0050] It will be appreciated that the methods of the present inventionand the use of a non-invasive or non-pathogenic bacterium as describedherein provide a wide range of therapeutic methods which would enablethe skilled person to manipulate, for instance, the immune response of asubject. Thus, the present invention provides, in various other aspects:

[0051] (i) a method of regulating the survival, growth, differentiation,effector functions or susceptibility to infection of cells or tissueswhich comprises administering to a subject a non-invasive ornon-pathogenic bacterium as defined herein;

[0052] (ii) a method of boosting an immune response against tumor cellsor an infection colonizing a mucosal surface or adjacent or distanttissue which comprises administering to a subject a non-invasive ornon-pathogenic bacterium as defined herein;

[0053] (iii) a method of modulating the type of immune response(antibody versus cell-mediated) against a pathogenic infectious agentwhich comprises administering to a subject a non-invasive ornon-pathogenic bacterium as defined herein;

[0054] (iv) a method of modulating the infiltration of normal tissueswith inflammatory or tumor cells which comprises administering to asubject a non-invasive or non-pathogenic bacterium as defined herein;

[0055] (v) a method of controlling the rate of growth, rate of invasionor survival of tumor cells which comprises administering to a subject anon-invasive or non-pathogenic bacterium as defined herein;

[0056] (vi) a method of inducing apoptosis in tumor cells whichcomprises administering to a subject a non-invasive or non-pathogenicbacterium as defined herein;

[0057] (vii) a method of downregulating an immune response whichcomprises administering to a subject a non-invasive or non-pathogenicbacterium which expresses a biologically active polypeptide; and

[0058] (viii) a method of treating an allergic autoimmune or otherimmune dysregulative disease state, which comprises administering to asubject a non-invasive or non-pathogenic bacterium which expresses abiologically active polypeptide.

[0059] Alternatively stated, when a cytokine and an antigen are bothexpressed by a bacterium, an aspect of the present invention provides amethod of enhancing an immune response to an antigen, the methodcomprising administering to an individual a non-invasive bacteriumcontaining nucleic acid for expression of a polypeptide with cytokineactivity and an antigen.

[0060] Enhancement of an immune response, such as an antibody response,preferably provides a level of immune response which is protective ofthe individual against subsequent challenge with the antigen in apathogenic context. For example, if the antigen is a bacterial toxoid ora toxin fragment, the level of an antibody response to administration ofa bacterium in accordance with the present invention may subsequentlyprotect the individual against pathogenic consequences of challenge withthe bacterial toxin, e.g., upon infection with bacteria which producethe toxin.

[0061] Administration of the bacterium by application to a mucosalsurface may be advantageous in certain contexts by virtue of generatingan enhanced immune response at the mucosal membrane (e.g., IgA response)in addition to a systemic response.

[0062] The bacterium may be applied in a nutrient medium, i.e., mediumcontaining a substance or substances which sustain (at least in vitro)metabolic activity in the bacterium. Such substances may sustainviability if not growth of the bacterium. Such substances may include anenergy source such as glucose, amino acids and so on.

[0063] The individual to which the bacterium is administered may behuman or animal, i.e., a non-human mammal. Administration mayconveniently be nasal, and may be oral, vaginal or anal. In contexts,where mucosal administration is not preferred, the bacterium may beadministered by any other suitable means within the capacity of thoseskilled in the art, e.g., by parental routes (i/v, i/p, s/c, i/m).

[0064] In a therapeutic context, i.e., where the biological effect ofdelivery of the polypeptide to an individual is beneficial to thatindividual, administration is preferably in a “therapeutically effectiveamount,” this being sufficient to show benefit to a patient. Suchbenefit may be at least amelioration of at least one symptom. In aprophylactic context, the amount may be sufficient to reduce thedeleterious effect on the individual of a subsequent pathogenicchallenge, for instance, by enhancing the immune response. The actualamount administered, and rate and time-course of administration, willdepend on the aim of the administration, e.g., the biological effectsought in view of the nature and severity of the challenge, and is thesubject of routine optimization. Prescription of treatment, includingprophylactic vaccination, for example, decisions on dosage, etc., iswithin the responsibility of general practitioners and other medicaldoctors.

[0065] A composition comprising bacteria may be administered inaccordance with the present invention alone or in combination with othertreatments, either simultaneously or sequentially.

[0066] The present invention also provides a pharmaceutical compositioncomprising a bacterium as disclosed. Such a pharmaceutical compositionis, in one embodiment, preferably suitable for application to a mucosalmembrane.

[0067] Pharmaceutical compositions according to the present invention,and for use in accordance with the present invention, may comprise, inaddition to the bacterium, a pharmaceutically acceptable excipient,carrier, buffer, stabilizer or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. The precise natureof the carrier or other material may depend on the route ofadministration. For intravenous, cutaneous or subcutaneous injection, orinjection at the site of an affliction, a parenterally acceptableaqueous solution may be employed which is pyrogen-free and has suitablepH, isotonicity and stability. Those of relevant skill in the art arewell able to prepare suitable solutions. Preservatives, stabilizers,buffers, antioxidants and/or other additives may be included, asrequired. As discussed, a pharmaceutical comprising a bacterium foradministration in accordance with the present invention may comprise oneor more nutrient substances, e.g., an energy source such as glucose,amino acids and so on.

[0068] In another aspect, the present invention provides a method ofmanufacture of a pharmaceutical comprising formulating bacteria asdisclosed with a suitable carrier medium for administration to anindividual. In one embodiment, the pharmaceutical is suitable forapplication to a mucosal membrane of an individual.

[0069] The present invention also provides a non-invasive bacteriumexpressing a heterologous biologically active polypeptide, and possiblyalso an antigen, for pharmaceutical use, i.e., use in a method oftreatment of the human or animal body by surgery or therapy, includingprophylaxis (“vaccination”). As disclosed, the bacterium may beGram-positive, is preferably non-commensal and/or is non-colonizing andsuitable examples include Lactococcus. The method preferably comprisesadministration to a mucosal membrane of an individual, e.g., to enhancean immune response in the individual.

[0070] A further aspect of the invention provides the use of anybacterium as disclosed in the manufacture of a composition, i.e., apharmaceutical composition or medicament, for administration to anindividual. Such administration is preferably to a mucosal membrane ofthe individual and may be to enhance an immune response in theindividual, e.g., to an antigen expressed by the bacterium.

[0071] Embodiments of each aspect of the present invention will beapparent from the disclosure and those skilled in the art willappreciate that modifications may be made. Further aspects andembodiments will be apparent. By way of experimental exemplification andnot limitation, use of an embodiment of the present invention inachieving a protective level of immune response to an antigen will nowbe described in detail with reference to the figures.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0072]FIG. 1 shows a flow scheme of plasmid constructions. The resultingplasmid pTTI2 may be used to express TTFC and IL-2, and resultingplasmid pTTI6 may be used to express TTFC and IL-6, in an organism suchas Lactococcus lactis.

[0073]FIG. 2a shows the vector pEX1 (also called pTREX1) into which agene, such as an operon construct comprising coding sequences for anantigen (e.g., TTFC) and a biologically active polypeptide (e.g., acytokine such as IL-2 or IL-6), may be inserted at the multiple cloningsite (MCS).

[0074]FIG. 2b shows an expanded view of a region of pEX1 (pTREX1)showing the P1 promoter, Shine-Dalgarno sequence (SD) and transcriptionterminator sequence operably positioned for expression of a gene(including a multi- (di-)cistronic coding sequence) when inserted at thegene MCS (multiple cloning site).

[0075]FIG. 3 shows the junction between the TTFC and Interleukincistrons in the operon employed for expression.

[0076]FIG. 4 shows TTFC-specific serum IgG titres of groups of six micevaccinated intranasally with recombinant Lactococcus lactis expressingtetanus toxin fragment C (TTFC) with the murine cytokines IL-2 or IL-6.

[0077] All documents mentioned herein are incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

[0078] To acquire the simultaneous expression of TTFC and either mIL2 ormIL6, we have chosen for the construction of operons driving the twocistrons under investigation. We made use of vectors for constitutiveexpression. In general, we try to flank cistrons with an XbaI siteimmediately prior to the Shine-Dalgarno (SD) sequence and an SpeI siteimmediately after the stop codon. In this way, multiple cistrons can beeasily exchanged and put in various combinations in any desired array,since XbaI and SpeI yield the same sticky ends. We have previouslyachieved the expression of mIL2 and mIL6 by means of the T7 promoter-T7gene 10 ribosome binding site, so we chose to use the XbaI site presentin the g10 ribosome binding site. For this arrangement, we knew the SDsequence was well positioned. We chose to put the TTFC cistron in frontof the interleukins.

[0079] Construction of Plasmids

[0080] The construction of the plasmids is depicted in FIG. 1. Plasmidscarrying mIL2 and mIL6 were subjected to site-directed mutagenesis togive extra SpeI sites immediately following the stop codons. Theresulting plasmids were called pL2MIL2A and pL2MIL6A, respectively. Aplasmid containing a fusion of the USP45 secretion leader and TTFC wasused as the template for PCR amplification of the various TTFC sequencesneeded.

[0081] For operons driving intracellular TTFC production, the gene wasamplified as a blunt—SpeI/BamHI fragment and cloned in the vectorpTREX1, which was cut with SphI, blunted and recut with BamHI. Theresulting plasmid was called pT1TT. From this plasmid, the 3′ terminal150 bp, SpeI TTFC fragment was isolated and cloned in the XbaI site ofpL2MIL2A and pL2MIL6A. The resulting plasmids were called p3TTIL2 andp3TTIL6. We made use of a KpnI restriction site present in the 3′ end ofTTFC to reconstruct TTFC and thus obtained the desired operons byligating the KpnI-SpeI fragment from p3TTIL2 and p3TTIL6 with theappropriate KpnI-PvuII and SpeI-PvuII fragments from pT1TT. Theresulting plasmids were called pTT12 and pTT16.

[0082] Expression of Proteins

[0083] The expression of proteins was assayed by antibody detection. Forthis, colonies of the different strains under investigation were spottedon nitrocellulose membranes and placed on GM17 (difco) solid agar platescontaining appropriate antibiotics. The plates were incubated overnightand blocked in PBS containing 2.5% skim milk powder. The filters wererevealed with rabbit-anti-TTFC or rabbit anti-MIL2. The experimentshowed clear TTFC expression in all constructs which hold the TTFC gene.Moreover, for pTTI2 and pTTAI2, the coexpression of IL2 and TTFC wasdetected. Since the junctions between TTFC units and mil6 are identicalto those between TTFC and mil2, it may be presumed that IL6 wascoexpressed with TTFC equally well.

[0084] Preparation of Cells for Immunizations

[0085] Bacterial strains for immunizations were grown from freshovernight cultures which were back diluted at a ratio of 1 ml ofovernight culture into 15 ml fresh GM17 medium containing erythromycinat 5 ug/ml and grown at 30° C. Cells were harvested at an opticaldensity at 600 nm of between 0.5 and 1.0. Cells were washed in {fraction(1/10)} of the original culture volume of 0.5% casamino acids, 0.2Msodium bicarbonate, and 0.5% glucose before resuspending in {fraction(1/200)}th of the original culture volume and determination of bacterialcell concentration. Cells were then diluted in the above solution togive the required number of cells per immunization.

[0086] Immunization

[0087] Mice were lightly anaesthetized by inhalation using “Metofane.”10 μl of the bacterial suspension, in a solution of 0.5% caseinhydrolysate, 0.2M sodium bicarbonate and 0.5% glucose, were applied toeach nostril in turn using an automatic pipette. The animals wereobserved closely for breathing difficulties until fully recovered fromanesthesia.

[0088] Results

[0089] Results are shown in Table 1 and FIG. 4. Bacteria able to expresseither IL-2 or IL-6 elicited 10× more anti-TTFC antibody than bacteriaexpressing TTFC alone.

[0090] It is the rule for bacterial toxins that a protective effect isachieved once the antibody titre exceeds a threshold value. The levelsof antibody titre found in the mice inoculated with bacteria containingpEX-TTFC/IL-2 and pEX-TTFC/IL-6 far exceeded the threshold value forsubsequent protection against tetanus toxin challenge (see FIG. 4,titres at 35 days post vaccination).

[0091] Summary of the Experimental Exemplification

[0092] Artificial operons for the coexpression of an antigenicpolypeptide (tetanus toxin fragment C-TTFC) and biologically activepolypeptides (IL-2; IL-6) were separately constructed in a constitutiveexpression vector (pTREX1) so that the transcription of the TTFC geneand the interleukin gene could be controlled by the activity of alactococcal promoter element of previously defined activity. Theconstructs were prepared so that, following translation of the mRNAtranscribed from the artificial operons, the TTFC antigen wouldaccumulate intracellularly. A secretion signal sequence was operablylinked to the interleukin. When preparations of these bacteria wereadministered intranasally to mice, bacteria engineered to express eitherIL-2 or IL-6 elicited approximately 10× more anti-TTFC antibody than theconstructs which expressed the TTFC alone. Thus, either of theseinterleukins possessed distinctive adjuvant activity in the experimentalsystem.

[0093]Lactococcus lactis is not a commensal bacterium (unlike relatedspecies of lactobacilli, which inhabit the crops of chickens and arepresent in the enteric tracts of many mammals) and also depends for itsnutrition on the provision of amino acids and peptides which areunlikely to be available in vivo, so the demonstration that thecytokine-expressing strains of L. lactis are nevertheless able toaugment antibody production is surprising. These results demonstrate forthe first time that recombinant strains of a non-colonizing,non-invasive bacterium such as Lactococcus lactis can be used tosynthesize and deliver biologically active molecules in vivo.

[0094] Table 1 (Overleaf)

[0095] In the table, “TT/9” is used to indicate inoculation withbacteria expressing TTFC at a dose of 1×10⁹ bacteria, “TT/8” at a doseof 1×10⁸ bacteria, and so on. “TT IL-2/9” and “TT IL-6/9” indicateinoculation with bacteria expressing TTFC and IL-2 and TTFC and IL-6,respectively, at a dose of 1×10⁹ bacteria, “TT IL-2/8” at a dose of1×10⁸ bacteria and so on. The figures given are ELISA titres forindividual mice. TABLE 1 IMMUNE RESPONSE DATA - DAY 35 End-point titresBleed 3 Nasal vaccinations data TT/9 TT/8 TT/7 TT/6 10000 50 50 50 1100060 50 50 10000 50 50 75 9000 50 50 50 4500 50 50 70 600 110 55 250 Mean7516.7 61.7 50.8 90.8 sd 4089.2 24.0 2.0 78.8 TT IL-2/9 TT IL-2/8 TTIL-2/7 TT IL-2/6 14000 50 50 50 30000 50 50 150 100000 50 50 50 100000105 150 50 120000 50 80 50 100000 100 50 50 Mean 77333.0 67.5 71.7 66.7sd 43848.0 27.2 40.2 40.8 TT IL-6/9 TT IL-6/8 TT IL-6/7 TT IL-6/6 80000200 50 50 170000 300 50 50 190000 200 100 50 100000 10000 50 50 50000750 50 50 80000 260 400 50 Mean 111670.0 1951.7 116.7 50.0 sd 55648.03948.3 140.2 0.0 CONTROLS pEX 1/9 pEX 1/8 pEX 1/7 pEX 1/6 Naïve 75 75 5575 60 75 50 75 55 60 50 55 75 75 55 55 50 55 50 55 75 55 75 50 50 60 5570 50 60 Mean 65.0 56.7 67.5 59.2 56.7 sd 11.4 0.9 1.0 12.4 4.1

[0096]

1 6 1 142 DNA Artificial Sequence P1 promoter derived from Lactococcuslactis 1 gattaagtca tcttacctct tttattagtt ttttcttata atctaatgataacattttta 60 taattaatct ataaaccata tccctctttg gaatcaaaat ttattatctactcctttgta 120 gatatgttat aatacaagta tc 142 2 81 DNA Artificial SequenceLeader Sequence of Usp45 in Lactococcus lactis 2 atg aaa aaa aag att atctca gct att tta atg tct aca gtg ata ctt 48 Met Lys Lys Lys Ile Ile SerAla Ile Leu Met Ser Thr Val Ile Leu 1 5 10 15 tct gct gca gcc ccg ttgtca ggt gtt tac gct 81 Ser Ala Ala Ala Pro Leu Ser Gly Val Tyr Ala 20 253 27 PRT Artificial Sequence Leader Sequence of Usp45 in Lactococcuslactis 3 Met Lys Lys Lys Ile Ile Ser Ala Ile Leu Met Ser Thr Val Ile Leu1 5 10 15 Ser Ala Ala Ala Pro Leu Ser Gly Val Tyr Ala 20 25 4 24 DNAArtificial Sequence pT1TT in Lactococcus lactis 4 acaaatgatt aaactagtggatcc 24 5 51 DNA Artificial Sequence pL2MIL2A, pL2MIL6A in Lactococcuslactis 5 tctagaaata attttgtttt actttaagaa ggagatatac atatgaaaaa a 51 663 DNA Artificial Sequence p3TTIL2, p3TTIL6, p3TTI2, pTTI6, inLactococcus lactis 6 acaaatgatt aaactagaaa taattttgtt ttactttaagaaggagatat acatatgaaa 60 aaa 63

What is claimed is:
 1. A method of delivering one or more antigens orbiologically active polypeptides to a subject in need of same whichcomprises administering to the subject a non-invasive or non-pathogenicbacterium which expresses the one or more antigens or polypeptides. 2.The method of claim 1 wherein the bacterium expresses both the one ormore antigens and one or more biologically active polypeptides.
 3. Themethod of claim 1 wherein the biologically active polypeptide isheterologous to the bacterium.
 4. The method of claim 3 wherein theheterologous polypeptide is derived from an eukaryote or its virus, froma prokaryote or its virus, or from a virus homologous to a species ofthe bacterium.
 5. The method of claim 1 wherein the bacterium is aGram-positive bacterium.
 6. The method of claim 5 wherein theGram-positive bacterium is Listeria innocua, Staphylococcus xylosus,Staphylococcus carnosus, Streptococcus gordoni, a Lactococcus species ora Lactobacillus species.
 7. The method of claim 6 wherein theGram-positive bacterium is Lactococcus lactis.
 8. The method of claim 5wherein the bacterium is an attenuated strain of a Gram-positivepathogenic bacterium.
 9. The method of claim 8 wherein the bacterium isListeria monocytogenes.
 10. The method of claim 1 wherein thebiologically active polypeptide is one which is capable of functioninglocally or systemically.
 11. The method of claim 1 wherein thebiologically active polypeptide is one which is capable of regulatingthe activities of cells belonging to the immunohemopoeitic system. 12.The method of claim 1 wherein the biologically active polypeptide is onewhich is capable of affecting the viability, growth and differentiationof normal or neoplastic cells in the body or affecting the immuneregulation or induction of acute phase inflammatory responses to injuryand infection.
 13. The method of claim 1 wherein the biologically activepolypeptide is one which is capable of enhancing or inducing resistanceto infection of cells and tissues mediated by chemokines acting on theirtarget cell receptors, or the proliferation of epithelial cells or thepromotion of wound healing.
 14. The method of claim 1 wherein thebiologically active polypeptide modulates the expression or productionof substances by cells in the body.
 15. The method of claim 1 whereinthe biologically active polypeptide is insulin, growth hormone,prolactin, calcitonin, luteinishing hormone, parathyroid hormone,somatostatin, thyroid stimulating hormone or vasoactive intestinalpolypeptide.
 16. The method of claim 1 wherein the biologically activepolypeptide is a structural group 1 cytokine adopting an antiparallel 4αhelical bundle structure such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-9, IL-10, IL-11, IL-12, IL-13, GM-CSF, M-CSF, SCR, IFN-γ, EPO, G-CSF,LIF, OSM, CNTF, GH, PRL or IFNα/β.
 17. The method of claim 1 wherein thebiologically active polypeptide is a structural group 2 cytokine whichforms symmetric homotrimers and the subunits take up the conformation ofjelly roll described for the TNF family of cytokines, the IL-1 family ofcytokines, the fibroblast growth factor family, the platelet derivedgrowth factors, transforming growth factor β or nerve growth factors.18. The method of claim 1 wherein the biologically active polypeptide isa structural group 3 cytokine comprising short chain α/β molecules,which are produced as transmembrane precursor molecules which eachcontain at least one EGF domain in the extracellular region.
 19. Themethod of claim 1 wherein the biologically active polypeptide is astructural group 4 cytokine which exhibits mosaic structures.
 20. Themethod of claim 1 wherein the biologically active polypeptide is areceptor or antagonist for a biologically active polypeptide.
 21. Themethod of claim 1 wherein the bacterium expresses an antigen orpolypeptide which regulates the survival, growth, differentiation,effector functions or susceptibility to infection of cells or tissues.22. The method of claim 1 wherein the bacterium expresses an antigen orpolypeptide which boosts an immune response against tumor cells or aninfection colonizing a mucosal surface or adjacent or distant tissue.23. The method of claim 1 wherein the bacterium expresses an antigen orpolypeptide which modulates the type of immune response (antibody versuscell-medicated) against a pathogenic infectious agent.
 24. The method ofclaim 1 wherein the bacterium expresses an antigen or polypeptide whichmodulates the infiltration of normal tissues with inflammatory or tumorcells.
 25. The method of claim 1 wherein the bacterium expresses anantigen or polypeptide which controls the rate of growth, rate ofinvasion or survival of tumor cells.
 26. The method of claim 1 whereinthe bacterium expresses an antigen or polypeptide which inducesapoptosis in tumor cells.
 27. The method of claim 1 wherein thebacterium expresses an antigen or polypeptide which downregulates animmune response.
 28. The method of claim 1, wherein the bacteriumexpresses an antigen or polypeptide which treats an allergic autoimmuneor immune dysregulative disease state.
 29. A pharmaceutical formulationcomprising a non-invasive or non-pathogenic bacterium having a nucleicacid construct or vector comprising one or more constitutive promotersand coding sequences for the expression of one or more antigens and/orheterologous biologically active polypeptides, and one or morepharmaceutically acceptable excipients, adjuvants, buffers, stabilizersor carriers.
 30. The pharmaceutical formulation of claim 29 as a vaccineformulation.
 31. A method of producing the pharmaceutical formulation ofclaim 29 which comprises the step of admixing one or more non-invasiveor non-pathogenic bacteria with one or more pharmaceutically acceptablecarriers.
 32. Nucleic acid comprising one or more coding sequences forone or more biologically active polypeptides and one or more codingsequences for one or more antigens wherein each coding sequence is underthe control of a promoter for expression in a non-invasive ornon-pathogenic bacterium.
 33. The nucleic acid of claim 32 whichcomprises one or more nucleic acid constructs in which the nucleic acidencoding the one or more biologically active polypeptides or antigensare under the control of appropriate regulatory sequences.
 34. Thenucleic acid of claim 33 wherein the appropriate regulatory sequencesare selected from promoter sequences, terminator fragments, enhancersequences or marker genes.
 35. The nucleic acid of claim 33 wherein theone or more nucleic acid constructs comprise an artificial operoncapable of generating a polycistronic RNA transcript.
 36. The nucleicacid of claim 32 wherein the promoter is a Lactococcal promoter for usein Lactococcus lactis.
 37. The nucleic acid of claim 32 which furthercomprises a secretory signal sequence, upstream of the codingsequence(s), for the one or more biologically active polypeptides. 38.The nucleic acid of claim 37 wherein the secretory signal sequene is theα-amylase secretion leader of Bacillus amyloliquefaciens, the secretionleader of the Staphylokinase enzyme, leader sequences for other Bacillusenzymes or S-layer proteins or the leader sequence of the Lactococcalprotein Usp45.
 39. The nucleic acid of claim 32 wherein the antigen isone capable of eliciting a protective immune response.
 40. The nucleicacid of claim 32 wherein the antigen is one where a protective immuneresponse is accelerated, amplified or rendered of longer duration in thepresence of one or more coexpressed biologically active polypeptides.41. A method of generating a bacterium which expresses one or moreantigens or biologically active polypeptides which comprises the step ofintroducing into a non-invasive or non-pathogenic bacterial host cellthe nucleic acid of claim
 32. 42. A non-invasive or non-pathogenicbacterium expressing (i) one or more heterologous biologically activepolypeptides and (ii) one or more antigens.
 43. A non-invasive ornon-pathogenic bacterium expressing (i) one or more heterologousbiologically active polypeptides and (ii) one or more antigens whichcomprises the nucleic acid of claim 32.